Achillea Millefolium L. Hydro- Alcoholic Extract Protects Pancreatic Cells by Down Regulating IL- 1β and iNOS Gene Expression in Diabetic Rats.

Interleukin-1β (IL-1β) has a role in β- cell destruction in autoimmune diabetes by stimulating the expression of inducible nitric oxide synthase (iNOS) that generates the free radical nitric oxide. We aimed to investigate the effect of Achillea millefolium L, as a traditional hypoglycemic agent, on IL-1β and iNOS gene expression of pancreatic tissue in the STZ- induced diabetic rats. Forty adult male Wistar rats were randomly divided into four groups: 1. diabetic control; 2. diabetic rats treated with Achillea millefolium L. extract; 3. normal rats received only extract and 4. negative control (n= 10 each). Diabetes was induced by single i.p. injection of 45 mg/ kg streptozotocin (STZ). Rats in groups 2 and 3 were treated with i.p. injection of Achillea millefolium L. extract (100 mg/ kg/ day) for 14 days. Body weight, serum glucose and insulin levels were assayed at baseline and on days 3, 7, 10 and 14 of the experiment. Finally, the quantity of pancreatic IL-1β and iNOS mRNA was determined by real- time PCR. The mRNA expression level of IL-1β and iNOS genes, was significantly (p<0.001) increased in diabetic rats of group 1. Treatment with Achillea millefolium L. caused a significant (p<0.01) reduction in both IL-1β and iNOS genes expression. Moreover, rats in group 2 had higher insulin level associated with lower glucose level and higher body weight compared to control diabetic group. It seems that beneficial effect of Achillea millefolium L. on STZ- induced diabetes is at least partly due to amelioration of IL-1β and iNOS gene over expression which can have a β-cell protective effect.

. Free radicals are formed disproportionately by glucose autoxidation, polyol pathway and non-enzymatic glycation of proteins in diabetes (6). Abnormal high levels of free radicals and simultaneous decline of antioxidant defense systems can lead to cellular damage, increased lipid peroxidation and development of diabetes mellitus complications (7).
Interleukin-1β (IL-1β) has been implicated as an effector molecule of β-cell destruction in autoimmune diabetes (8,9). This cytokine is increased in patients with newly diagnosed T1D and likely acts as an early inflammatory signal in T1D development (10). It is possible that IL-1β acts in concert with other cytokines to cause islet cell death and it has been demonstrated that the cytotoxic effect of IL-1β is associated with the expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide (11).
Several Achillea species are used for their pharmaceutical, cosmetic and fragrance properties.
Their extracts exhibit pharmacological activities such as anti-inflammatory, analgesic and antipyretic. Achillea millefolium L. (yarrow) has shown a wide range of therapeutic applications such as treating wounds, colds, fevers, kidney diseases, stopping blood flow and reducing menstrual pain (12). It has traditionally been used as a hypoglycemic agent in diabetic status (13)(14)(15), however the mechanism(s) behind its hypoglycemic effect are not fully elucidated, thus this study aimed to investigate the effect of Achillea millefolium L.
on IL-1β and iNOS gene expression of pancreatic tissue in the STZ-induced diabetic rats.

Plant material
Aerial parts of the plant were collected from a local store and identified by the Department of The SYBR Green RT-PCR assay was carried out as previously described in detail (18). The results for IL-1β mRNA levels were presented relative to the expression of GAPDH (a housekeeping gene).

Statistical analysis
Data were presented as mean± SD. Data analysis was carried out by using one-way ANOVA and Tukey's multiple comparison test as the post hoc (SPSS 11.5 for windows software).
Differences were considered significant at p<0.05.  3 remained statistically the same as negative control during the experiment ( Table 1).

Diabetic
The mRNA expression level of IL-1β and iNOS genes assessed by real-time PCR, was significantly (P<0.001) increased in diabetic rats of group 1 as compared to negative control. Treatment with Achillea millefolium L. caused a significant (P<0.01) reduction in both IL-1β and iNOS genes expression (about 56 and 55 percent, respectively).
The IL-1β/ GAPDH mRNA ratio was 3.76 fold higher in diabetic rats compared with that in control ones and treatment with extract reduced the ratio to 1.92 fold (Fig. 1). The ratio of iNOS/ GAPDH mRNA was 3 fold greater in diabetic rats as compared with that in control ones and was reduced to 1.76 fold after treatment with extract (Fig. 2).  Achillea millefolium L. extract ( Fig.1 and 2).

Discussion
Traditional plant treatments have been used all over the world for diabetes (19). In recent years, T1D is believed to be an immune-mediated process in which pro-inflammatory cytokines particularly IL-1β plays an essential role (23,24).
Furthermore, the role of IL-1β in T2D has also been established (25,26). IL-1 β has been implicated in early events in β-cell destruction.
Suppression of IL-1β production or inhibition of its interaction with corresponding cellular receptors significantly inhibits IL-1β-mediated deleterious effects on β-cells (27,28) and direct blockade of IL-1β has been studied as a therapeutic strategy for T1D at the preclinical level (10). In vitro observations indicate that the cytotoxic effect of IL-1β in islet cells involves the induction of nitric oxide synthase (iNOS) and the production of nitric oxide (29,30). Several flavonoids have been shown to inhibit the expression of NF-κB-dependent cytokines, iNOS, and cyclooxygenase-2 genes (39). This needs to be further evaluated in future studies.
In conclusion it seems that beneficial effect of Achillea millefolium L. on STZ-induced diabetes is at least partly due to amelioration of IL-1β and iNOS gene over expression which can have a β-cell protective effect.